CN110938125A - Creation and application of high molecular weight glutenin subunit mutant gene 1Dx2m1 for improving wheat processing quality - Google Patents

Creation and application of high molecular weight glutenin subunit mutant gene 1Dx2m1 for improving wheat processing quality Download PDF

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CN110938125A
CN110938125A CN201911292331.6A CN201911292331A CN110938125A CN 110938125 A CN110938125 A CN 110938125A CN 201911292331 A CN201911292331 A CN 201911292331A CN 110938125 A CN110938125 A CN 110938125A
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wheat
gln
1dx2m1
flour
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张相岐
张鲁军
范仁春
卫波
孙家柱
高彩霞
张爱民
王道文
贾旭
王献平
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Institute of Genetics and Developmental Biology of CAS
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Abstract

The invention discloses a method for creating a high molecular weight glutenin subunit mutant gene 1Dx2m1 for improving the processing quality of wheat and application thereof. The invention adopts a large primer PCR mutation technology to carry out site-directed mutagenesis on the HMW-GS gene 1Dx2 of wheat, so that the 353 rd base C of the coding region of the HMW-GS gene is mutated into G, and the single base mutant gene 1Dx2m1 is obtained. By transforming the mutant gene 1Dx2m1 into the 1Dx2 subunit deletion mutant kn2 of the common wheat variety Kenong 199‑And a series of processing quality parameter analysis proves that: the mutant high molecular weight glutenin subunit 1Dx2m1 can significantly improve the processing quality of wheat flour. The invention relates to the site-directed mutagenesis of HMW-GS gene 1Dx2 and the transformationThe gene method improves the processing quality of wheat, fills the blank of improving the processing quality of wheat by using point mutant genes at home and abroad, and opens up a new way for improving the processing quality of wheat.

Description

Creation and application of high molecular weight glutenin subunit mutant gene 1Dx2m1 for improving wheat processing quality
Technical Field
The invention belongs to the technical field of biology, and particularly relates to creation and application of a high molecular weight glutenin subunit mutant gene 1Dx2m1 for improving the processing quality of wheat.
Background
Common wheat (Triticum aestivum l., AABBDD, 2n ═ 6x ═ 42) belongs to the gramineous (Poaceae) wheat (Triticum) genus, and is the most important food crop for human beings. Because the wheat dough has unique elasticity and ductility, the wheat dough can be processed into various foods such as bread, biscuits, noodles, steamed bread, dumplings, fried bread sticks and the like through the processes of baking, cooking, frying and the like. The quality of the food is closely related to the quality of the wheat. Wheat quality refers to the degree to which a wheat variety adapts and meets a particular end use and can be divided into nutritional quality and processing quality. The nutritional quality refers to the degree of satisfaction of nutritional ingredients contained in wheat to human (livestock) nutritional needs, including the amount of nutritional ingredients, whether the nutritional ingredients are balanced, the content of protein, the composition of amino acids, and the like. The processing quality is divided into primary processing quality and secondary processing quality. The quality of primary processing is also called grinding quality, and mainly comprises flour yield, flour color, ash content and the like. The secondary processing quality, also called eating quality, refers to the degree of adaptation and satisfaction of wheat flour to the preparation of different foods, and is mainly related to the physicochemical properties of the dough. The edible quality parameters are many and mainly comprise gluten content, sedimentation value, forming time, stabilizing time, resistance to elongation, stretching area and specific indexes aiming at various baked, steamed and fried wheaten foods.
Currently, the quality improvement of wheat is mainly focused on the processing quality, and is also mainly focused on the baking quality of industrially processed bread, biscuits, cakes and the like, and the baking quality mainly depends on the gluten content and quality of flour. After the flour is mixed with water and subjected to a suitable kneading, a dough is formed, the dough is washed with a suitable concentration of weak saline water to remove bran, starch and proteins dissolved in water or dilute salt, and the remaining material having a viscoelastic network structure is called gluten (gluten), which consists of glutenin and gliadin. The properties of gluten determine the rheological properties of the dough and thus the food processing quality of the wheat variety. In many studies, gluten strength is often used to describe the food processing quality of wheat, and wheat can be classified into strong gluten wheat, medium gluten wheat and weak gluten wheat according to the strength of gluten. The method for measuring and identifying the quality of wheat mainly comprises physicochemical detection (including using various special instruments and analysis methods) and food processing detection (including baking, cooking and the like). The main indexes for measuring the processing quality of the wheat flour comprise gluten content, sedimentation value, water absorption, forming time, stabilization time, resistance to elongation, stretching area, swelling index, various kneading and mixing instrument parameters and the like.
SDS-sedimentation value (SDS-SV) is a comprehensive index reflecting wheat gluten content and quality, and is deeply valued by wheat quality breeding workers. The principle is that a certain amount of wheat flour absorbs water to expand under the action of weak acid medium to form floccule and slowly precipitate, and the sedimentation speed and volume reflect the content and quality of gluten. The sedimentation volume in the specified standard time is the sedimentation value, and is taken as the unit of ml. The larger the measured value, the higher the gluten content, the higher the gluten strength, and the better the baking quality of the flour. The Swelling Index (SIG) of glutenin is an important index for wheat quality evaluation, and is in obvious positive correlation with the SDS-sedimentation value. SIG indirectly reflects the content and quality of glutenin based on the swelling capacity of glutenin at different swelling times. SIG measurement has the characteristics of rapidness, simplicity, convenience, small sample consumption and the like, and is often used for quality detection of early-generation materials in wheat quality breeding.
The Farinograph (Farinograph) is one of the most widely used wheat quality detection instruments, and can be used for quality detection and quality control of wheat grains or flour in the processes of wheat breeding, storage, flour processing, food production and the like. The flour quality instrument is used for kneading by adding a proper amount of water into quantitative wheat flour, and a coordinate graph of the change of stirring resistance along with time, namely a flour quality curve, is drawn by computer software. And calculating the quality characteristic parameters of the flour according to the water adding amount and the flour quality curve so as to evaluate the strength of the gluten of the flour. The main parameters measured by the flour quality meter are Water Absorption (WA), dough formation time (DT), dough Stability Time (ST), degree of weakness (DS), and Flour Quality Number (FQN). WA is affected by factors such as protein content and starch granule breakage rate in wheat flour, and the higher the protein content and starch granule breakage rate, the higher the water absorption rate. DT and ST reflect the stability and degree of dough resistance. The longer DT and ST of the dough, indicating greater gluten strength, generally better bake processing characteristics. DS represents the rate of dough breakdown during mixing, reflecting the strength of the gluten. The larger the DS, the weaker the gluten, the more rheological the dough, the less processable and the less good the baking quality. FQN is an important index reflecting rheological characteristics of dough, and has a close relation with gluten strength, the FQN of strong gluten flour is higher, and the FQN of weak gluten flour is lower.
The mixer (mixogrph) is an important instrument for measuring the quality of wheat processing by measuring the change in rheological properties during the stirring of the dough. In the continuous kneading process of the dough, the kneading instrument measures and records the kneading resistance of the dough and draws a kneading curve map to provide the optimal kneading time, stirring endurance, rheological characteristics of other dough, baking estimated water absorption value and other indexes of the dough. The kneading and mixing instrument has the advantages of high detection speed, small flour consumption, high correlation with baking characteristics and the like, and has good guiding function on wheat quality improvement, wheat quality chemical research and development of special flour and flour products. Among the parameters obtained by the kneading apparatus, the Middle Peak Time (MPT) is a very important parameter. Generally, it is considered that the MPT has high correlation with the kneading time of actual and experimental baking and is an important prediction index of bread quality. The midline peak height (MPV) is related to the extensibility of the dough and the toasted volume of the bread, the Midline Peak Width (MPW) is related to the stretch resistance of the dough, and the midline peak area (MPI) is significantly related to the maximum stretch resistance of the dough, the stretch area, and the toasted volume. The fade resistance value (RBD) is related to the kneading resistance of the dough, with the greater the RBD, the weaker the strength of the dough and the poorer the kneading resistance.
Disclosure of Invention
The first object of the present invention is to provide a protein obtained by mutating serine at position 118 of the amino acid sequence of 1Dx2 protein to cysteine, which is designated as 1Dx2m1 protein.
The 1Dx2 protein is a protein shown in a) or b) as follows:
a) the amino acid sequence is a protein shown in a sequence 4;
b) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 4.
The 1Dx2m1 protein is a protein shown in the following c) or d):
c) the amino acid sequence is a protein shown in a sequence 8;
d) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 8.
In the above protein, the tag is a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.
It is a second object of the present invention to provide a biomaterial related to the 1Dx2m1 protein.
The biomaterial is any one of the following A1) to A8):
A1) a nucleic acid molecule encoding a 1Dx2m1 protein;
A2) an expression cassette comprising the nucleic acid molecule of a 1);
A3) a recombinant vector comprising the nucleic acid molecule of a 1);
A4) a recombinant vector comprising the expression cassette of a 2);
A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);
A6) a recombinant microorganism comprising the expression cassette of a 2);
A7) a recombinant microorganism comprising a3) said recombinant vector;
A8) a recombinant microorganism comprising the recombinant vector of a 4).
In the above biological material, the nucleic acid molecule of A1) is a gene represented by the following 1) or 2) or 3):
1) the coding sequence is a cDNA molecule or a DNA molecule shown in a sequence 7;
2) a cDNA molecule or a genome DNA molecule which has 95 percent or more than 95 percent of identity with the nucleotide sequence defined by 1) and codes 1Dx2m1 protein;
3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes for 1Dx2m1 protein.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
The nucleotide sequence of the present invention encoding 1Dx2m1 can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence encoding 1Dx2m1 are derived from and identical to the nucleotide sequence of the present invention as long as they encode 1Dx2m1 and have the same function.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 8 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.
In the above biological material, the stringent conditions are hybridization and membrane washing at 68 ℃ for 2 times, 5min each, in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing at 68 ℃ for 2 times, 15min each, in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.
In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The recombinant vector can be specifically a recombinant vector p1Bx14PRO-1Dx2m 1.
In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.
The third purpose of the invention is to provide a new application of the 1Dx2m1 protein or biological material.
The invention provides an application of the 1Dx2m1 protein or the biological material in any one of the following B1) -B10):
B1) regulating and controlling the processing quality of the wheat flour;
B2) regulating and controlling the baking quality of the wheat flour;
B3) regulating and controlling the gluten property and/or gluten strength and/or gluten quality of the wheat flour;
B4) regulating the dough rheological property and/or the extensibility and/or the stretch resistance and/or the kneading resistance of the wheat flour;
B5) regulating and controlling the SDS-sedimentation value of the wheat flour;
B6) regulating and controlling the glutenin swelling index of the wheat flour;
B7) regulating and controlling the water absorption and/or the dough forming time and/or the dough stabilizing time and/or the weakening degree in the flour quality instrument detection parameters of the wheat flour;
B8) regulating and controlling peak time and/or peak area and/or anti-fading value in the parameters of the kneading and mixing instrument of the wheat flour;
B9) cultivating transgenic wheat with improved flour processing quality and/or baking quality and/or gluten quality;
B10) and (5) wheat breeding.
In the application, the regulation and control of the processing quality of the wheat flour is specifically to improve the processing quality of the wheat flour;
the method is characterized in that the baking quality of the wheat flour is regulated and controlled, and specifically the baking quality of the wheat flour is improved;
the gluten property and/or the gluten strength and/or the gluten quality of the wheat flour are regulated and controlled, and specifically the gluten property of the wheat flour is improved and/or the gluten strength of the wheat flour is improved and/or the gluten quality of the wheat flour is improved;
the dough rheological property and/or the extensibility and/or the stretch resistance and/or the kneading resistance of the wheat flour are/is regulated, and particularly the dough rheological property and/or the extensibility and/or the stretch resistance and/or the kneading resistance of the wheat flour are improved;
the SDS-sedimentation value of the wheat flour is specifically adjusted and controlled to be improved;
the glutenin swelling index of the wheat flour is regulated and controlled, and is specifically the glutenin swelling index of the wheat flour is improved;
the method for regulating and controlling the water absorption and/or the dough forming time and/or the dough stabilizing time and/or the weakening degree in the flour quality instrument detection parameters of the wheat flour is to specifically improve the water absorption in the flour quality instrument detection parameters of the wheat flour and/or improve the dough forming time in the flour quality instrument detection parameters of the wheat flour and/or improve the dough stabilizing time in the flour quality instrument detection parameters of the wheat flour and/or reduce the weakening degree in the flour quality instrument detection parameters of the wheat flour;
the peak time and/or the peak area and/or the anti-fading value in the detection parameters of the kneading and mixing instrument for regulating and controlling the wheat flour are/is specifically to improve the peak time in the detection parameters of the kneading and mixing instrument for regulating and controlling the wheat flour and/or improve the peak area in the detection parameters of the kneading and mixing instrument for regulating and controlling the wheat flour and/or reduce the anti-fading value in the detection parameters of the kneading and mixing instrument for regulating and controlling the wheat flour;
the aim of the wheat breeding is to improve the processing quality and/or baking quality and/or gluten quality of wheat flour.
A fourth object of the present invention is to provide a method for breeding transgenic wheat having improved flour processing quality and/or baking quality and/or gluten quality.
The method for cultivating the transgenic wheat with improved flour processing quality and/or baking quality and/or gluten quality comprises the steps of improving the content and/or activity of 1Dx2m1 protein in receptor wheat to obtain transgenic wheat; the transgenic wheat has a higher flour processing quality and/or baking quality and/or gluten quality than the recipient wheat.
In the above method, the transgenic wheat has higher flour processing quality than the recipient wheat as embodied in any one of C1) -C9):
C1) the SDS-sedimentation value of the transgenic wheat flour is higher than that of receptor wheat;
C2) the swelling index of glutenin of the transgenic wheat flour is higher than that of receptor wheat;
C3) the water absorption rate in the detection parameters of the flour quality instrument of the transgenic wheat flour is higher than that of receptor wheat;
C4) the dough forming time in the flour quality instrument detection parameters of the transgenic wheat flour is longer than that of receptor wheat;
C5) the dough stability time in the flour quality instrument detection parameters of the transgenic wheat flour is longer than that of receptor wheat;
C6) the attenuation degree of the detection parameters of the flour quality instrument of the transgenic wheat flour is lower than that of receptor wheat;
C7) the peak time in the detection parameters of the kneading and mixing instrument of the transgenic wheat flour is higher than that of receptor wheat;
C8) the peak value area in the detection parameters of the kneading and mixing instrument of the transgenic wheat flour is higher than that of receptor wheat;
C9) the anti-fading value of the parameters detected by the kneading and mixing instrument of the transgenic wheat flour is lower than that of receptor wheat.
Further, the method for improving the content and/or activity of the 1Dx2m1 protein in the receptor wheat is to over-express the 1Dx2m1 protein in the receptor wheat.
Furthermore, the method for over-expressing the 1Dx2m1 protein in the recipient wheat is to introduce a coding gene of the 1Dx2m1 protein into the recipient wheat. The nucleotide sequence of the coding gene of the 1Dx2m1 protein is specifically shown as a DNA molecule shown as a sequence 7.
In a specific embodiment of the invention, the gene encoding the 1Dx2m1 protein is introduced into recipient wheat through a recombinant vector p1Bx14PRO-1Dx2m 1. The recombinant vector p1Bx14PRO-1Dx2m1 is a vector obtained by replacing a sequence 13 in a sequence table with a vector pUBI, wherein the DNA fragment is positioned between the Pst I and Kpn I enzyme cutting sites of cas, and other sequence structures are kept unchanged.
In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the 1Dx2m1 gene into a plant of interest, but also its progeny. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.
In the method, the receptor wheat is a 1Dx2 subunit deletion mutant (kn 2) of wheat elite Kenong 199-Mutant). The kn2-The mutant is a 1Dx2 deletion mutant obtained by deleting only 1Dx2 gene (sequence 3) in the genome sequence of the common wheat variety Kenong 199(KN 199).
The invention adopts a large primer PCR mutation technology to carry out site-directed mutagenesis on the HMW-GS gene 1Dx2 of wheat to obtain a single-site mutant gene 1Dx2m1, the mutant gene 1Dx2m1 can be translated into a mutant high molecular weight glutenin subunit 1Dx2m1 in vivo, and a gene gun mediated method is adopted to introduce the mutant gene 1Dx2m1 into a 1Dx2 subunit deletion mutant of a good variety of wheat, i.e. farmer 199, so as to obtain a stable transgenic line TL1Dx2m 1. Quality analysis experiments prove that the mutant high molecular weight glutenin subunit 1Dx2m1 can obviously improve the processing quality of wheat flour. The invention improves the processing quality of wheat by site-directed mutagenesis of HMW-GS gene 1Dx2 and a transgenic method, fills the blank of improving the processing quality of wheat by using the site-directed mutagenesis gene at home and abroad, and opens up a new way for improving the processing quality of wheat.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products of the full-length sequence of the coding region of 1Dx2 gene. Note: lane M is DNAmarker; the KN199 Lane is a product obtained by performing PCR amplification by using a specific primer combination Dx2F and Dx2R for amplifying the full-length sequence of the coding region of the 1Dx2 gene and taking the genome DNA of the common wheat variety Kenong 199 as a template, and the band marked by 1Dx2 is an amplified fragment of the full-length sequence of the coding region of the 1Dx2 gene.
FIG. 2 is a schematic diagram of the mutation site of 1Dx2m1 gene. Note: SP is a signal peptide sequence; NT is an N-terminal sequence; CRD is the middle repeat region; CT is C-terminal sequence; m1 is 1Dx2m1 site-directed mutation site, namely 353 rd base C is mutated into base G.
FIG. 3 is a schematic diagram showing the process for obtaining 1Dx2m1 gene. Note: a is the amplification of a Dx2FRm1 site-directed mutagenesis large primer; b is the amplification of the full-length sequence of the coding region of the 1Dx2m1 gene. Lane M is DNA marker; lane 1Dx2 shows PCR amplification products of the coding region of the 1Dx2 gene using primer combinations Dx2F and Dx2m1R (A) and Dx2m1FR and Dx2R (B), respectively, as templates.
FIG. 4 shows the general wheat variety Kenong 199 and its 1Dx2 subunit deletion line kn2-SDS-PAGE of glutenin. Note: kn2-1Dx2 subunit deletion line for konong 199; KN199 is wild type Kenong 199; the right side labeled 1Dx2 etc. are the corresponding subunits.
FIG. 5 shows the construction process of 1Dx2m1 gene transformation vector p1Bx14PRO-1Dx2m 1.
FIG. 6 is T0PCR detection of generation 1Dx2m1 transgenic wheat plants. Note: m is DNA marker, KN is wild type Kenong 199, KN is transgenic receptor, namely 1Dx2 subunit deletion mutant KN2 of Kenong 199-And B is blank control. The 414bp band indicated by the arrow is the target amplified fragment, + represents a positive plant, -represents a negative plant.
FIG. 7 is T3SDS-PAGE detection of the generation 1Dx2m1 transgenic line HMW-GS. Note: kn2-Is a transgenic receptor, namely a deletion mutant of 1Dx2 subunit of Kenong 199; KN199 is wild type Kenong 199; TL1Dx2 is a 1Dx2 transgenic line; TL1Dx2m1 is a transgenic line for the mutant gene 1Dx2m 1.
FIG. 8 shows the 1Dx2m1 transgenic lines TL1Dx2m1, TL1Dx2 transgenic line TL1Dx2 and transgenic receptor kn2-And a powdery picture of wild type Kenong 199(KN 199).
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The powder quality instrument (Farinograph-E) in the following examples is a product of Brabender, Ohgduisburg, Germany.
The kneading apparatus (mixogrph) in the following examples is a product of National company (National mfg.co., Lincoln, USA).
The vector pAHC20 containing the selection marker gene Bar in the following examples is a product of Pubaezin Biotechnology (Beijing) Ltd.
The general wheat cultivar kong 199 in the following examples was bred by Lijunming researcher of institute of genetics and developmental biology, national institute of sciences, and the cultivar was examined and approved: national scrutiny wheat 2006017.
Example 1 obtaining of wheat mutant Gene 1Dx2m1
1. PCR amplification of wheat 1Dx2 gene coding region full-length sequence
The genome DNA of the general wheat variety Ke nong 199 is used as a template, and PCR amplification is carried out by using a specific primer combination Dx2F and Dx2R, high fidelity PrimeSTAR HS DNA polymerase and 2 XPrimeSTAR GC Buffer (Takara, large connection) to obtain a PCR amplification product (figure 1), namely the full-length sequence of the coding region of the 1Dx2 gene. The nucleotide sequences of the specific primer combinations Dx2F and Dx2R amplified by PCR are respectively sequence 1 and sequence 2 in the sequence table.
PCR amplification is carried out by a two-step method. The reaction procedure is as follows: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 deg.C for 30sec, renaturation at 72 deg.C and extension for 3min, and circulation for 38 times; finally, extension is carried out for 10min at 72 ℃.
2. Recovery and clone sequencing of 1Dx2 gene PCR amplification product
After the PCR amplification product was separated by 1% agarose gel electrophoresis, a gel portion containing a target band (about 2500bp) was cut under an ultraviolet lamp, and placed in a 1.5ml centrifuge tube, and the amplified DNA fragment was recovered using an agarose gel recovery kit (Tiangen, Beijing). The recovered fragment was ligated to the Blunt-end cloning vector pEasy blast (all-trans gold, Beijing) and sequenced (Huada gene, Beijing). The sequencing result shows that the length of the cloned DNA fragment is 2523bp, and the nucleotide sequence is the sequence 3 in the sequence table, namely the full-length sequence of the coding region of the 1Dx2 gene. 1Dx2 is a gene without intron, and the coded 1Dx2 protein consists of 839 amino acid residues, and the amino acid sequence of the protein is the sequence 4 in the sequence table.
3. Obtaining of Single base mutant Gene 1Dx2m1 of 1Dx2
Site-directed mutagenesis is carried out on m1 site of 1Dx2 by adopting a large primer PCR method to obtain single base mutant gene 1Dx2m 1. The single-base mutation is a mutation of 353 rd cytosine (C) to guanine (G) in the coding region sequence of 1Dx2 gene. A schematic of the mutation sites is shown in FIG. 2. The specific operation steps are as follows:
(1) using the full-length sequence (sequence 3) of the coding region of 1Dx2 gene as a template, PCR amplification was performed using PrimeSTAR HS DNA polymerase, 2 × PrimeSTAR GC Buffer (precious bio, large ligation) and primer combination Dx2F and Dx2m1R of the amplified primer sequence to obtain a PCR product (fig. 3A). The nucleotide sequences of the primer combination Dx2F and Dx2m1R of the amplified primer sequence are respectively sequence 1 and sequence 5 in the sequence table.
The PCR reaction procedure for amplifying the large primer sequence employs a two-step process. The specific procedure is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30sec, annealing at 72 ℃ and extension for 1min for 30 cycles; extension at 72 ℃ for 10 min.
(2) After the PCR product is identified by 1% agarose gel electrophoresis, a large primer fragment containing a mutation site is obtained by recovery sequencing and is named as Dx2m1 FR. The size of the large primer fragment Dx2m1FR is 364bp, and the nucleotide sequence thereof is the sequence 6 in the sequence table.
(3) The full-length sequence (sequence 3) of the coding region of the 1Dx2 gene was used as a template, and PCR amplification was performed using a large primer set Dx2m1FR (sequence 6) and Dx2R (sequence 2) to obtain a PCR product (FIG. 3B).
(4) And (3) separating, recovering, cloning and sequencing the PCR product (shown in figure 3B) obtained in the step (3) to obtain the full-length coding region sequence of the single-base mutant gene 1Dx2m1 of 1Dx 2. The full length of the coding region sequence of 1Dx2m1 is 2523bp, and the nucleotide sequence is the sequence 7 in the sequence table. In fact, the coding region sequence of the 1Dx2m1 gene is a nucleotide sequence which changes the 353 rd base cytosine (C) of the coding region sequence of the 1Dx2 gene shown in the sequence 3 in the sequence table into guanine (G) and keeps other sequences unchanged. The protein subunit coded by the mutant gene 1Dx2m1 is HMW-GS with single amino acid residue mutation, and is named as 1Dx2m1, 1Dx2m1 is composed of 839 amino acid residues, and the amino acid sequence is sequence 8 in the sequence table. In fact, the amino acid sequence of the mutant subunit 1Dx2m1 is an amino acid sequence obtained by mutating the serine residue (Ser) at the 118 th position of the 1Dx2 subunit shown in the sequence 4 into the cysteine residue (Cys) and keeping other sequences unchanged.
Example 2, 1Dx2m1 obtaining of transgenic wheat
1. Acquisition of transgenic recipient Material
The transgenic receptor material used by the invention is a high molecular weight glutenin subunit 1Dx2 deletion mutant of common wheat variety Kenong 199(KN199), which is marked as KN2-。kn2-Mutants were obtained by EMS mutagenesis. The specific process is as follows: (1) 1000 filled wild type farmer 199 seeds were selected and treated with 0.4% EMS (Sigma, M0880) in the dark for 24 hours and washed with running water for 24 hours, and the seeds were marked M0；(2)M0The seeds are planted in the Beijing Changping experiment base of the institute of genetics and development biology of Chinese academy of sciences after normal germination and vernalization treatment (4 ℃) for 4 weeks, normal field management is carried out, and the harvested seeds are recorded as M1(ii) a From M by SDS-PAGE1Screening out 1Dx2 subunit deletion mutant from seeds, and marking as kn2-(FIG. 4); m1Generation kn2-The seeds are backcrossed and selfed for two successive generations to obtain enough M3Generation seeds as the recipient material for genetic transformation of the mutant gene 1Dx2m 1. Sequencing verification shows that: kn2-The mutant is a 1Dx2 deletion mutant obtained by deleting only 1Dx2 gene (sequence 3) in the genome sequence of the common wheat variety Kenong 199(KN199), and the rest sequences are the same as the genome sequence of the common wheat variety Kenong 199(KN 199).
2. Construction of transformation vectors for mutant Gene 1Dx2m1 and wild-type Gene 1Dx2
The construction process of the 1Dx2m1 gene transformation vector is shown in FIG. 5. The specific operation steps are as follows:
(1) taking the genome DNA of common wheat variety Elytrigia tritici 54 as a template, utilizing primer combination Bx14proF and Bx14proR to carry out PCR amplification to obtain an 1147bp 1Bx14 promoter sequence, and simultaneously adding recognition sequences of PstI and BamH I restriction endonucleases at the upstream and downstream of the sequence respectively. The nucleotide sequences of the primer combination Bx14proF and Bx14proR are respectively a sequence 9 and a sequence 10 in a sequence table. The obtained 1Bx14 promoter sequence is the sequence 11 in the sequence table.
(2) And (2) inserting the 1Bx14 promoter sequence (sequence 11 in a sequence table) obtained in the step (1) into a vector pUBI: (sequence 12 in the sequence table) between Pst I and BamH I enzyme digestion sites of cas to replace the Ubiquitin promoter sequence, and keeping other sequences of the vector pUBI: (sequence 12 in the sequence table) unchanged to obtain an intermediate vector p1Bx14 PRO.
(3) The single base mutant gene 1Dx2m1 coding region sequence (sequence 7 in the sequence table) is inserted between BamH I and Kpn I enzyme cutting sites of intermediate vector p1Bx14PRO, namely connected to the downstream of 1Bx14 promoter sequence, and other sequences of intermediate vector p1Bx14PRO are kept unchanged, so that recombinant vector p1Bx14PRO-1Dx2m1 is obtained, namely the transformation vector of 1Dx2m1 gene.
(4) Sequencing verification is carried out on the recombinant vector p1Bx14PRO-1Dx2m1 to ensure that the sequence inserted into the recombinant vector p1Bx14PRO-1Dx2m1 is a sequence (sequence 13 in a sequence table) formed by connecting a 1Bx14 promoter and a 1Dx2m1 coding region, namely the recombinant vector p1Bx14PRO-1Dx2m1 is a vector obtained by replacing the sequence 13 in the sequence table with a DNA fragment between the enzyme cutting sites Pst I and Kpn I of cas, and keeping other sequence structures unchanged.
The construction process and operation procedure of the wild-type 1Dx2 gene transformation vector are the same as those of the mutant gene 1Dx2m1 transformation vector, except that the coding region sequence of the wild-type 1Dx2 (sequence 3 in the sequence table) is used to replace the coding region sequence of the mutant gene 1Dx2m1 (sequence 7 in the sequence table). The transformation vector was designated p1Bx14PRO-1Dx 2.
3. Genetic transformation of the 1Dx2m1 Gene
Genetic transformation was performed by the particle gun method. The transformation acceptor material is 1Dx2 subunit deletion mutation system kn2 of excellent common wheat variety Kenong 199-Young embryos about 14 days after flowering. Co-transforming the transformation vector p1Bx14PRO-1Dx2m1 with a vector pAHC20 containing a selection marker gene Bar to obtain T of the mutation-transferred gene 1Dx2m10And (5) plant generation. Genetic transformation is carried out on the genetic transformation platform of institute of genetics and developmental biology of the Chinese academy of sciences. The genetic transformation of the wild-type 1Dx2 gene was performed in the same manner as the genetic transformation of the mutant gene 1Dx2m1 except that the transformation vector was p1Bx14PRO-1Dx 2.
4. PCR identification and propagation of 1Dx2m1 gene transformant
T of transgenic 1Dx2m1 Using primer combination Dx2nosF and Dx2nosR0Performing PCR identification on the generation plants by using a receptor kn2-Is a negative control. If the PCR amplification product is a 414bp fragment (FIG. 6), the plant is a positive plant of the transmutant gene 1Dx2m1, and if the 414bp amplification fragment does not exist, the plant is a negative plant. The sequences of the primers Dx2nosF and Dx2nosR are respectively sequence 14 and sequence 15 in the sequence table. Identification of wild-type 1Dx2 Gene transformant T was detected by SDS-PAGE0The method of generation seed glutenin subunit, the plant expressing 1Dx2 subunit is the positive plant of 1Dx2 gene.
Positive plants transformed with 1Dx2m1 were planted and the seeds were harvested individually. 3 transgenic 1Dx2m1 wheat homozygous lines with stable expression are obtained by PCR of continuous 3-4 generations of exogenous genes and SDS-PAGE identification of HMW-GS (figure 7), and are respectively marked as TL1Dx2m1-5, TL1Dx2m1-14 and TL1Dx2m1-15, and are used for determining processing quality traits. Seeds of plants positive for the transgene 1Dx2 were also propagated as controls for the analysis of the processing quality of the transgenic line with the mutant gene 1Dx2m 1.
Example 3, determination of processing quality traits in 1Dx2m1 transgenic lines and comparative analysis thereof with recipient, wild type Kenong 199 and 1Dx2 transgenic lines
The invention provides for two consecutive years of 1T obtained in example 2 in 2016 and 2017, respectively3Generation 1Dx2m1 transgenic line TL1Dx2m1 and 2T4The generation 1Dx2m1 transgenic lines TL1Dx2m1-5 and TL1Dx2m1-15 are subjected to processing quality trait determination and are matched with a transgenic receptor kn2-The 1Dx2 transgenic line TL1Dx2 (1 line in 2016, 2 lines in 2017) and wild type konnong 199 were compared. All assay samples were assigned 3 biological replicates. Data collection, processing and comparative analysis were performed using Excel (Microsoft, USA) software, One-way ANOVA (One-way ANOVA) and multiple comparisons using SPSS19.0(IBM, USA) software, and multiple comparisons using the Duncon method in One-way ANOVA.
1. Determination and analysis of SDS-sedimentation values
The SDS sedimentation value (SDS-SV) of the 1Dx2m1 transgenic line was determined in 2016 and 2017, respectively, according to the method described in "Zhang L., Chen Q., Su M., Yan B., Zhang X., Jiano Z., high-molecular-weight genetic dilution with mutation induced by beam and their effects of Glu-1loci deletion on heat quality properties. J.Sci.FoodAgr.,2016,96: 1289-1296". The specific operation steps are as follows: weighing 1g flour (14% water content, and the non-14% water content sample is converted to 14% water content) in a 35ml glass test tube with plug scale, adding 16.8ml bromophenol blue aqueous solution (4mg/L), and mixing by reverse for 10-20 sec; placing the test tube on CAU-B sedimentation value analyzer (China university of agriculture), and shaking for 5min for 40 times/min; adding 16.8ml of SDS-lactic acid solution (2% SDS, 0.19% lactic acid), reversing, mixing, placing on a sedimentation value analyzer, and shaking for 5min for 40 times/min; and (5) reversing and mixing for 10-20sec, standing for 5min, and observing and recording sedimentation value results. The transgenic receptor kn2 is used in the experiment-The transgenic line TL1Dx2 of the wild type gene 1DX2 and wild type Kenong 199 were controls.
The results of the SDS sedimentation value measurement are shown in Table 1. As can be seen from table 1: in the year 2016, the number of years,the SDS-sedimentation value of the transgenic line TL1Dx2m1 is obviously greater than that of a transgenic receptor kn2-1DX2 transgenic line TL1Dx2 and wild type KN 199. In 2017, the SDS-sedimentation value of 21 Dx2m1 transgenic lines TL1Dx2m1-5 and TL1Dx2m1-15 is also not only significantly larger than that of transgenic receptor kn2-And also significantly greater than the 1DX2 transgenic line TL1DX2 and wild-type KN 199. The mutant subunit 1Dx2m1 is proved to be capable of obviously improving the SDS sedimentation value of the flour compared with the wild subunit 1Dx 2.
TABLE 1 SDS-sedimentation values of 1Dx2m1 transgenic lines
Figure BDA0002319574690000091
Note: (1) the data in the table are the mean of the measurements for two consecutive years of seed (2016 and 2017); (2) different letters after the measurement represent significant differences at the 0.05 level.
2. Determination and analysis of glutenin swelling index SIG
The glutenin Swelling index (Swelling index of glutenin, SIG) of the 1Dx2m1 transgenic line was determined in 2016 and 2017, respectively, and likewise in kn2, according to the method described in "Wang, C., and Kovacs, M.I.P.spinning index of glutenintest.II.application in prediction of dough properties and end-use quality. Central chem.J.2002,79:190--TL1Dx2 and wild type Kenong 199 as controls. All assay samples were assigned 3 biological replicates. The specific operation steps are as follows: (1) 50ml of the tubes were weighed individually (M0), and 1.0g of flour of the transgenic line was added to each tube and weighed (M1). (2) 15ml of distilled water was added to the centrifuge tube, vortexed for 10sec, and allowed to stand at 24 ℃ for 20min, during which vortexed for 2 times for 10 sec. (3) 15ml of SDS-lactic acid solution (3% SDS, 0.19% lactic acid) was added to each centrifuge tube, vortexed for 10sec, and allowed to stand at 24 ℃ for hydration for 20min, during which vortexed for 2 times for 10 sec. (4) Centrifuging at 450 Xg for 5min, taking out the centrifuge tube, removing most of supernatant by pipette without touching interface, centrifuging the precipitate at 450 Xg for 3min, immediately inverting the centrifuge tube on water-absorbing filter paper, removing residual water on the tube wall one by one, and weighing (M)2). (5) The weight of wet batter formed per weight of flour (M2-M0). times.100/(M1-M0). times.100-W, where W is the moisture content of the sample, was calculated as the SIG value.
The SIG measurement results are shown in Table 2. As can be seen from Table 2, in 2016, the SIG value of transgenic line TL1Dx2m1 was significantly greater than that of transgenic receptor kn2-1DX2 transgenic line TL1Dx2 and wild type KN 199. In 2017, the SIG values of 21 Dx2m1 transgenic lines TL1Dx2m1-5 and TL1Dx2m1-15 are also not only significantly higher than that of transgenic receptor kn2-And are also both significantly higher than the transgenic line TL1Dx2 and wild type Konong 199. The over-expression of the mutant subunit 1Dx2m1 is proved to be capable of obviously improving the swelling index of glutenin, and the effect of the subunit is obviously better than that of the wild type 1Dx2 subunit.
TABLE 2 glutenin Swelling Index (SIG) of 1Dx2m1 transgenic lines
Figure BDA0002319574690000092
Figure BDA0002319574690000101
Note: (1) the data in the table are the mean of the measurements for two consecutive years of seed (2016 and 2017); (2) different letters after the measurement represent significant differences at the 0.05 level.
3. Powder analysis
According to American society of cereal chemistry, AACCthSt.MN, USA American Association of Central Chemistry,2000 "and the program" AACC 54-21 "were each subjected to flour quality analysis on flour of transgenic lines expressing the mutant gene 1Dx2m 1. The experiment was repeated 3 times using a 50g dough bowl. The specific operation steps are as follows: (1) TL1Dx2m1 flour (14% wet basis, 50g) was weighed according to the moisture content of the flour according to the data given by the instrument software. After the instrument is opened and zero calibration is carried out, the flour is put into a dough kneading pot of the flour quality instrument, and a dough kneading pot cover is covered. (2) Starting the dough kneading device switch, stirring and kneading for 1min, rapidly adding water, and covering with a dough kneading pot cover. After the dough is kneaded until the curve is peaked, whether the peak value of the curve is 5 or not is observed00 +/-20 FU. (3) If the peak value is between 500 +/-20 FU, continuing kneading dough, continuing softening for 12min after the curve starts to obviously decrease, finishing the powder quality map, and performing data acquisition and comparative analysis.
The main parameters of the powder quality testing are shown in table 3. As can be seen from Table 3, through 2016 and 2017 continuous two-year determination, various indexes (except the water absorption index of 2017) of the powder quality instrument of the transgenic line TL1Dx2m1 are remarkably superior to those of a transgenic receptor kn2-Transgenic lines TL1Dx2 and wild-type KN 199. The dough formation time and the stability time of the transgenic line TL1Dx2m1 are also significantly better than that of the receptor kn2-Transgenic lines TL1Dx2 and wild-type KN199 (fig. 8). The mutant gene 1Dx2m1 is shown to be capable of improving the rheological property of wheat dough obviously.
TABLE 3 powder quality instrument parameters of 1Dx2m1 transgenic line
Figure BDA0002319574690000102
Note: (1) the data in the table are the mean of the measurements for two consecutive years of seed (2016 and 2017); (2) different letters after the measurement represent significant differences at the 0.05 level.
4. Kneading and mixing instrument analysis
The dough peak time (MPT), peak height (V), peak width (MPW), peak area (MPI) and anti-fade value (RBD) of the 1Dx2m1 transgenic line were determined in 2016 and 2017, respectively, using a kneader according to the methods of documents "AACC 54-40A" and "Sun et al (2015) (Sun, J., Yang, W., Liu, D., ZHao, J., Luo, G., Li, X., Liu, Y., Guo, J., and Zhang, A. (2015). Improv on Mixograph third water addition and parameter conversion.J.Integr.Agr.14: 1715-1722.). The method comprises the following specific steps: a kneading bowl of 10g was used. The total kneading time of the standard kneading machine is 10min, and the weighing amount M of the flour is (10g × 86)/(100-W), wherein W is the water content of the sample. The data obtained were analyzed with MixSmart software (AEW Consulting, Lincoln, Nebraska, USA). The curve of the kneading and mixing instrument is automatically drawn by a computer, the parameters exceed 40, and the analysis usually adopts the middle line (Midline) parameters which are divided into 4 categories of belt height, bandwidth, area and slope. Each index of the kneading and mixing instrument spectrum reflects the rheological characteristics and quality of the wheat dough.
The results of the measurement of the main parameters of the kneading apparatus are shown in Table 4. As can be seen from table 4: in 2016, both the dough peak time and peak area were significantly higher than the control for the 1Dx2m1 transgenic line TL1Dx2m1, while the fade resistance was significantly lower than the control. The peak heights and peak widths were not significantly or irregularly different compared to the control. In 2017, the kneading and mixing parameters of TL1Dx2m1-5 and TL1Dx2m1-15 of 2 transgenic lines were consistent with those of 2016. It is shown that mutant subunit 1Dx2m1 can improve the rheological properties of dough.
TABLE 4 kneading and mixing parameters for 1Dx2m1 transgenic lines
Figure BDA0002319574690000111
The SDS-sedimentation value (SDS-SV) and the gluten Swelling Index (SIG) are comprehensive indexes for evaluating the quality and quantity of wheat gluten, and are highly positively correlated with the gluten strength. The peak time (MPT) and the peak area (MPI) in the parameters of the kneading and mixing device, the forming time and the Stable Time (ST) in the parameters of the flour quality device, the flour quality index (FQN) and the gluten quality are positively correlated, and the anti-fading value (RBD) in the parameters of the kneading and mixing device and the weakening Degree (DS) in the parameters of the flour quality device are negatively correlated with the gluten quality.
The experimental results of the above two years are combined to show that: the SDS, SIG, MPT, MPI and FQN of the transgenic line expressing the mutant gene 1Dx2m1 are all obviously higher than those of a control (transgenic receptor kn 2)-1Dx2 transgenic line and wild type Kenong 199), and control (transgenic receptor kn 2)-1Dx2 transgenic line and wild type konong 199), has longer formation time and stabilization time, and simultaneously has lower RBD and DS, so the TL1Dx2m1 transgenic line has better gluten quality, thereby showing that the mutant gene 1Dx2m1 can obviously improve the gluten characteristics of wheat flour and improve the processing quality.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of genetics and developmental biology of Chinese academy of sciences
<120> creation and application of high molecular weight glutenin subunit mutant gene 1Dx2m1 for improving wheat processing quality
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Met Ala Lys Arg Leu Val Leu Phe Val Ala Val Val Val Ala Leu Val
1 5 10 15
Ala Leu Thr Val Ala Glu Gly Glu Ala Ser Glu Gln Leu Gln Cys Glu
20 25 30
Arg Glu Leu Gln Glu Leu Gln Glu Arg Glu Leu Lys Ala Cys Gln Gln
35 40 45
Val Met Asp Gln Gln Leu Arg Asp Ile Ser Pro Glu Cys His Pro Val
50 55 60
Val Val Ser Pro Val Ala Gly Gln Tyr Glu Gln Gln Ile Val Val Pro
65 70 75 80
Pro Lys Gly Gly Ser Phe Tyr Pro Gly Glu Thr Thr Pro Pro Gln Gln
85 90 95
Leu Gln Gln Arg Ile Phe Trp Gly Ile Pro Ala Leu Leu Lys Arg Tyr
100 105 110
Tyr Pro Ser Val Thr Ser Pro Gln Gln Val Ser Tyr Tyr Pro Gly Gln
115 120 125
Ala Ser Pro Gln Arg Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln
130 135 140
Gln Ser Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Pro Gln Gln
145 150 155 160
Pro Gly Gln Trp Gln Gln Pro Glu Gln Gly Gln Pro Gly Tyr Tyr Pro
165 170 175
Thr Ser Pro Gln Gln Pro Gly Gln Leu Gln Gln Pro Ala Gln Gly Gln
180 185 190
Gln Pro Gly Gln Gly Gln Gln Gly Arg Gln Pro Gly Gln Gly Gln Pro
195 200 205
Gly Tyr Tyr Pro Thr Ser Ser Gln Leu Gln Pro Gly Gln Leu Gln Gln
210 215 220
Pro Ala Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly
225 230 235 240
Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln
245 250 255
Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln Gln
260 265 270
Leu Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Ser
275 280 285
Gly Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Leu Gly
290 295 300
Gln Gly Gln Ser Gly Tyr Tyr Pro Thr Ser Pro Gln Gln Pro Gly Gln
305 310 315 320
Gly Gln Gln Pro Gly Gln Leu Gln Gln Pro Ala Gln Gly Gln Gln Pro
325 330 335
Glu Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln
340 345 350
Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Pro Gly Tyr Tyr
355 360 365
Pro Thr Ser Pro Gln Gln Ser Gly Gln Gly Gln Pro Gly Tyr Tyr Pro
370 375 380
Thr Ser Ser Gln Gln Pro Thr Gln Ser Gln Gln Pro Gly Gln Gly Gln
385 390 395 400
Gln Gly Gln Gln Val Gly Gln Gly Gln Gln Ala Gln Gln Pro Gly Gln
405 410 415
Gly Gln Gln Pro Gly Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Pro
420 425 430
Leu Gln Ser Gly Gln Gly Gln Pro Gly Tyr Tyr Leu Thr Ser Pro Gln
435 440 445
Gln Ser Gly Gln Gly Gln Gln Pro Gly Gln Leu Gln Gln Ser Ala Gln
450 455 460
Gly Gln Lys Gly Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly
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Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln Gln Pro Gly
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Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Pro Gln Gln Ser Gly Gln
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Gly Gln Gln Pro Gly Gln Trp Gln Gln Pro Gly Gln Gly Gln Pro Gly
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Tyr Tyr Pro Thr Ser Pro Leu Gln Pro Gly Gln Gly Gln Pro Gly Tyr
530 535 540
Asp Pro Thr Ser Pro Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln
545 550 555 560
Leu Gln Gln Pro Ala Gln Gly Gln Gln Gly Gln Gln Leu Ala Gln Gly
565 570 575
Gln Gln Gly Gln Gln Pro Ala Gln Val Gln Gln Gly Gln Gln Pro Ala
580 585 590
Gln Gly Gln Gln Gly Gln Gln Leu Gly Gln Gly Gln Gln Gly Gln Gln
595 600 605
Pro Gly Gln Gly Gln Gln Pro Ala Gln Gly Gln Gln Gly Gln Gln Pro
610 615 620
Gly Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly
625 630 635 640
Gln Gly Gln Pro Trp Tyr Tyr Pro Thr Ser Pro Gln Glu Ser Gly Gln
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Gly Gln Gln Pro Gly Gln Trp Gln Gln Pro Gly Gln Trp Gln Gln Pro
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Gly Gln Gly Gln Pro Gly Tyr Tyr Leu Thr Ser Pro Leu Gln Leu Gly
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Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Pro Gly Gln
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Gly Gln Gln Pro Gly Gln Trp Gln Gln Ser Gly Gln Gly Gln His Gly
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Tyr Tyr Pro Thr Ser Pro Gln Leu Ser Gly Gln Gly Gln Arg Pro Gly
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Gln Trp Leu Gln Pro Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser
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Pro Gln Gln Ser Gly Gln Gly Gln Gln Leu Gly Gln Trp Leu Gln Pro
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Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Thr Gly
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Gln Gly Gln Gln Ser Gly Gln Gly Gln Gln Gly Tyr Tyr Ser Ser Tyr
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His Val Ser Val Glu His Gln Ala Ala Ser Leu Lys Val Ala Lys Ala
805 810 815
Gln Gln Leu Ala Ala Gln Leu Pro Ala Met Cys Arg Leu Glu Gly Gly
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Asp Ala Leu Ser Ala Ser Gln
835
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<213>Artificial Sequence
<400>5
cctgctgcgg acaagttaca cttgggtaa 29
<210>6
<211>364
<212>DNA
<213>Artificial Sequence
<400>6
atggctaagc ggttagtcct ctttgtggcg gtagtcgtcg ccctcgtggc tctcaccgtc 60
gctgaaggtg aggcctctga gcaactacag tgtgagcgcg agctccagga gctccaggag 120
cgcgagctca aggcatgcca gcaggtcatg gaccagcagc tccgagacat tagccccgag 180
tgccaccccg tcgtcgtcag cccggtcgcg ggacaatacg agcagcaaat cgtggtgccg 240
cccaagggcg gatctttcta ccccggcgag accacgccac cgcagcaact ccaacaacgt 300
atattttggg gaatacctgc actactaaaa aggtattacc caagtgtaac ttgtccgcag 360
cagg 364
<210>7
<211>2523
<212>DNA
<213>Artificial Sequence
<400>7
atggctaagc ggttagtcct ctttgtggcg gtagtcgtcg ccctcgtggc tctcaccgtc 60
gctgaaggtg aggcctctga gcaactacag tgtgagcgcg agctccagga gctccaggag 120
cgcgagctca aggcatgcca gcaggtcatg gaccagcagc tccgagacat tagccccgag 180
tgccaccccg tcgtcgtcag cccggtcgcg ggacaatacg agcagcaaat cgtggtgccg 240
cccaagggcg gatctttcta ccccggcgag accacgccac cgcagcaact ccaacaacgt 300
atattttggg gaatacctgc actactaaaa aggtattacc caagtgtaac ttgtccgcag 360
caggtttcat actatccagg ccaagcttct ccgcaacggc caggacaagg tcagcagcca 420
ggacaagggc aacaatcagg acaaggacaa caagggtact acccaacttc tccgcaacag 480
ccaggacaat ggcaacaacc ggaacaaggg caaccagggt actacccaac ttctccgcag 540
cagccaggac aattgcaaca accagcacaa gggcagcaac caggacaagg acaacaaggt 600
cggcagccag gacaagggca accagggtac tacccaactt cttcgcagct gcagccagga 660
caattgcaac aaccagcaca agggcaacaa gggcagcaac caggacaagg gcaacaaggt 720
caacagccag gacaagggca acaaccagga caaggacaac aaggtcaaca gccaggacaa 780
gggcaacaac caggacaagg gcaacaaggt cagcagctcg gacaaggaca acaagggtac 840
tacccaactt ctctgcaaca gtcgggacaa gggcaaccag ggtactaccc aacttctctg 900
cagcagctag gacaagggca atcagggtac tacccaactt ctccgcagca accaggacaa 960
gggcagcagc caggacaatt gcaacaacca gcacaagggc agcaaccaga acaagggcaa 1020
caaggtcagc agccaggaca agggcaacaa ggccagcagc caggacaagg gcagcaaccg 1080
ggacaagggc aaccagggta ctacccaact tctccgcagc agtcaggaca agggcaacca 1140
gggtactacc caacttcttc gcagcagcca acacaatcgc agcaaccagg acaagggcaa 1200
caaggtcagc aggtaggaca agggcaacaa gctcagcagc caggacaagg gcagcaaccg 1260
ggacaagggc agccagggta ctacccaact tctccgctgc agtcaggaca agggcaacca 1320
gggtactacc taacttctcc gcagcagtca ggacaagggc agcagccagg acaattgcaa 1380
caatcagcac aagggcaaaa aggacagcaa ccaggacaag gtcaacagcc agggcaaggg 1440
caacaaggtc agcagccagg acaagggcaa caaggtcagc aaccggggca agggcagcca 1500
gggtactacc caacttctcc gcagcaatca ggacaagggc aacagccagg acaatggcaa 1560
caaccaggac aagggcaacc aggatactac ccaacttctc cgttgcagcc aggacaaggg 1620
caaccagggt acgacccaac ttctccgcaa cagccaggac aagggcagca accaggacaa 1680
ttgcaacaac cagcacaagg gcaacaaggg cagcaactag cacaagggca acaagggcag 1740
caaccagcac aagtgcaaca agggcagcag ccagcacaag ggcaacaagg tcagcagcta 1800
ggacaagggc aacaaggtca gcagccagga caagggcagc aaccagcaca agggcaacaa 1860
ggtcagcagc caggacaagg gcaacaaggt cagcagccag gacaagggca gcaaccggga 1920
caagggcagc catggtacta cccaacttct ccgcaggagt caggacaagg gcaacagcca 1980
ggacaatggc aacaaccagg acaatggcaa caaccaggac aagggcaacc agggtactac 2040
ctaacttctc cgttgcagct aggacaaggg caacaagggt actacccaac ttctctgcaa 2100
caaccaggac aagggcagca accaggacaa tggcaacaat cgggacaagg gcaacatggg 2160
tactacccaa cttctccgca gctgtcagga caagggcaac ggccaggaca atggctgcaa 2220
ccaggacaag ggcaacaagg gtactaccca acttctccgc aacagtcagg acaagggcaa 2280
caactaggac aatggctgca accaggacaa gggcaacaag ggtactaccc aacttctctg 2340
caacagacag gacaagggca gcaatcagga caagggcaac aaggctacta cagctcatac 2400
catgttagcg tggagcacca ggcggccagc ctaaaggtgg caaaggcgca gcagctcgcg 2460
gcacagctgc cggcaatgtg ccggctggag ggcggcgacg cattgtcggc cagccagtga 2520
tag 2523
<210>8
<211>839
<212>PRT
<213>Artificial Sequence
<400>8
Met Ala Lys Arg Leu Val Leu Phe Val Ala Val Val Val Ala Leu Val
1 5 10 15
Ala Leu Thr Val Ala Glu Gly Glu Ala Ser Glu Gln Leu Gln Cys Glu
20 25 30
Arg Glu Leu Gln Glu Leu Gln Glu Arg Glu Leu Lys Ala Cys Gln Gln
35 4045
Val Met Asp Gln Gln Leu Arg Asp Ile Ser Pro Glu Cys His Pro Val
50 55 60
Val Val Ser Pro Val Ala Gly Gln Tyr Glu Gln Gln Ile Val Val Pro
65 70 75 80
Pro Lys Gly Gly Ser Phe Tyr Pro Gly Glu Thr Thr Pro Pro Gln Gln
85 90 95
Leu Gln Gln Arg Ile Phe Trp Gly Ile Pro Ala Leu Leu Lys Arg Tyr
100 105 110
Tyr Pro Ser Val Thr Cys Pro Gln Gln Val Ser Tyr Tyr Pro Gly Gln
115 120 125
Ala Ser Pro Gln Arg Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln
130 135 140
Gln Ser Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Pro Gln Gln
145 150 155 160
Pro Gly Gln Trp Gln Gln Pro Glu Gln Gly Gln Pro Gly Tyr Tyr Pro
165 170 175
Thr Ser Pro Gln Gln Pro Gly Gln Leu Gln Gln Pro Ala Gln Gly Gln
180 185 190
Gln Pro Gly Gln Gly Gln Gln Gly Arg Gln Pro Gly Gln Gly Gln Pro
195 200 205
Gly Tyr Tyr Pro Thr Ser Ser Gln Leu Gln Pro Gly Gln Leu Gln Gln
210 215 220
Pro Ala Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly
225 230 235 240
Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln
245 250 255
Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln Gln
260 265 270
Leu Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Ser
275 280 285
Gly Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Leu Gly
290 295 300
Gln Gly Gln Ser Gly Tyr Tyr Pro Thr Ser Pro Gln Gln Pro Gly Gln
305 310 315 320
Gly Gln Gln Pro Gly Gln Leu Gln Gln Pro Ala Gln Gly Gln Gln Pro
325 330 335
Glu Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln
340 345 350
Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Pro Gly Tyr Tyr
355 360 365
Pro Thr Ser Pro Gln Gln Ser Gly Gln Gly Gln Pro Gly Tyr Tyr Pro
370 375 380
Thr Ser Ser Gln Gln Pro Thr Gln Ser Gln Gln Pro Gly Gln Gly Gln
385 390 395 400
Gln Gly Gln Gln Val Gly Gln Gly Gln Gln Ala Gln Gln Pro Gly Gln
405 410 415
Gly Gln Gln Pro Gly Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Pro
420 425 430
Leu Gln Ser Gly Gln Gly Gln Pro Gly Tyr Tyr Leu Thr Ser Pro Gln
435 440 445
Gln Ser Gly Gln Gly Gln Gln Pro Gly Gln Leu Gln Gln Ser Ala Gln
450 455 460
Gly Gln Lys Gly Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly
465 470 475 480
Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln Gln Pro Gly
485 490 495
Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Pro Gln Gln Ser Gly Gln
500 505 510
Gly Gln Gln Pro Gly Gln Trp Gln Gln Pro Gly Gln Gly Gln Pro Gly
515 520 525
Tyr Tyr Pro Thr Ser Pro Leu Gln Pro Gly Gln Gly Gln Pro Gly Tyr
530 535 540
Asp Pro Thr Ser Pro Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln
545 550 555 560
Leu Gln Gln Pro Ala Gln Gly Gln Gln Gly Gln Gln Leu Ala Gln Gly
565 570 575
Gln Gln Gly Gln Gln Pro Ala Gln Val Gln Gln Gly Gln Gln Pro Ala
580 585 590
Gln Gly Gln Gln Gly Gln Gln Leu Gly Gln Gly Gln Gln Gly Gln Gln
595 600 605
Pro Gly Gln Gly Gln Gln Pro Ala Gln Gly Gln Gln Gly Gln Gln Pro
610 615 620
Gly Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly
625 630 635 640
Gln Gly Gln Pro Trp Tyr Tyr Pro Thr Ser Pro Gln Glu Ser Gly Gln
645 650 655
Gly Gln Gln Pro Gly Gln Trp Gln Gln Pro Gly Gln Trp Gln Gln Pro
660 665 670
Gly Gln Gly Gln Pro Gly Tyr Tyr Leu Thr Ser Pro Leu Gln Leu Gly
675 680 685
Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Pro Gly Gln
690 695 700
Gly Gln Gln Pro Gly Gln Trp Gln Gln Ser Gly Gln Gly Gln His Gly
705 710 715 720
Tyr Tyr Pro Thr Ser Pro Gln Leu Ser Gly Gln Gly Gln Arg Pro Gly
725 730 735
Gln Trp Leu Gln Pro Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser
740 745 750
Pro Gln Gln Ser Gly Gln Gly Gln Gln Leu Gly Gln Trp Leu Gln Pro
755 760 765
Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Thr Gly
770 775 780
Gln Gly Gln Gln Ser Gly Gln Gly Gln Gln Gly Tyr Tyr Ser Ser Tyr
785 790 795 800
His Val Ser Val Glu His Gln Ala Ala Ser Leu Lys Val Ala Lys Ala
805 810 815
Gln Gln Leu Ala Ala Gln Leu Pro Ala Met Cys Arg Leu Glu Gly Gly
820 825 830
Asp Ala Leu Ser Ala Ser Gln
835
<210>9
<211>33
<212>DNA
<213>Artificial Sequence
<400>9
aactgcagag ggaaagacaa tggacatgca aag 33
<210>10
<211>33
<212>DNA
<213>Artificial Sequence
<400>10
cgggatccct cggtgaactg tcagtgaatt gat 33
<210>11
<211>1147
<212>DNA
<213>Artificial Sequence
<400>11
agggaaagac aatggacatg caaagaggta ggggcaggga agaaacactt ggagatcata 60
gaagaacata agaggttaaa cataggagca gtggcggagc ttgggctgat tttatggagg 120
ggcaaatggg ctggaggggc aagaaatatg agtttgggct gattttaact gggcatatgg 180
gctgaatact aggggatata tgctagtttt ctcatgggct gggggggcaa tggcccaggt 240
tgctctccac taagctccgc cactgcatag gagggcataa tggacaatta aatctacatt 300
aattgaactc atttgggaag taaacaaaat ccatattctg gtgtaaatca aactatttga 360
tgcggattta ctaagatcct atgttaattt tagacatgac tggccaaagg tttcagttag 420
ttcatttgtc acggaaaggt gttttcataa gtccaaaact ctaccaactt ttttgcacgt 480
catagcatag atagatgttg tgagtcattg gatagatatt gtgagtcagc atggatttgt 540
gttgcctgga aatccaacta aatgacaagc aacaaaacct gaaatgggct ttaggagaga 600
tggtttatca atttacatgt tccatgcagg ctaccttcca ctactcgaca tggttagaag 660
ttttgagtgc cgcatatttg cggaagcaat ggcactactc gacatggtta gaagttttga 720
gtgccgcata tttgcggaag caatggctaa cagatacata ttctgccaaa ccccaagaag 780
gataatcact cctcttagat aaaaagaaca gaccaatgta caaacatcca cacttctgca 840
aacaatacac cagaactagg attaagccca ttacgtggct ttagcagacc gtccaaaaat 900
ctgttttgca agcaccaatt gctccttact tatccagctt cttttgtgtt ggcaaactgc 960
ccttttccaa ccgattttgt tcttctcacg ctttcttcat aggctaaact aacctcggcg 1020
tgcacacaac catgtcctga accttcacct cgtccctata aaagcccatc caaccttcac 1080
aatctcatca tcacccacaa caccgagcac cccaatctac agatcaattc actgacagtt 1140
caccgag 1147
<210>12
<211>4691
<212>DNA
<213>Artificial Sequence
<400>12
gaaccctgca gtgcagcgtg acccggtcgt gcccctctct agagataatg agcattgcat 60
gtctaagtta taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt 120
atctatcttt atacatatat ttaaacttta ctctacgaat aatataatct atagtactac 180
aataatatca gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca 240
attgagtatt ttgacaacag gactctacag ttttatcttt ttagtgtgca tgtgttctcc 300
tttttttttg caaatagctt cacctatata atacttcatc cattttatta gtacatccat 360
ttagggttta gggttaatgg tttttataga ctaatttttt tagtacatct attttattct 420
attttagcct ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaataattt 480
agatataaaa tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt 540
aaaaaaacta aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc 600
gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa 660
gcagacggca cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc 720
gttggacttg ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc 780
ggcacggcag gcggcctcct cctcctctca cggcacggca gctacggggg attcctttcc 840
caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 900
ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 960
tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 1020
taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 1080
atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 1140
cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 1200
ctgggatggc tctagccgtt ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt 1260
gcatagggtt tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg 1320
ggtcatcttt tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc 1380
gttctagatc ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc 1440
tgtatgtgtg tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg 1500
atctaggata ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt 1560
tgttcgcttg gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg 1620
agtagaatac tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg 1680
tcatacatct tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat 1740
acatgttgat gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat 1800
atgctctaac cttgagtacc tatctattat aataaacaag tatgttttat aattattttg 1860
atcttgatat acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg 1920
ccttcatacg ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt 1980
tggtgttact tctgcaggtc gactctagag gatccccggg taccgagctc gaatttcccc 2040
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 2100
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 2160
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 2220
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 2280
atgttactag atcgggaatt catcgatgat atcagatctg ccggtctccc tatagtgagt 2340
cgtattaatt tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag 2400
aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 2460
cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 2520
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 2580
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 2640
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 2700
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 2760
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 2820
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 2880
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 2940
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 3000
tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 3060
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 3120
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 3180
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 3240
aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 3300
tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 3360
cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 3420
catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 3480
ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 3540
aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 3600
ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 3660
caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 3720
attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 3780
agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 3840
actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 3900
ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 3960
ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 4020
gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 4080
atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 4140
cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 4200
gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 4260
gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 4320
ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 4380
gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga 4440
tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 4500
ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 4560
ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt 4620
gtcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag 4680
gtgacactat a 4691
<210>13
<211>3676
<212>DNA
<213>Artificial Sequence
<400>13
agggaaagac aatggacatg caaagaggta ggggcaggga agaaacactt ggagatcata 60
gaagaacata agaggttaaa cataggagca gtggcggagc ttgggctgat tttatggagg 120
ggcaaatggg ctggaggggc aagaaatatg agtttgggct gattttaact gggcatatgg 180
gctgaatact aggggatata tgctagtttt ctcatgggct gggggggcaa tggcccaggt 240
tgctctccac taagctccgc cactgcatag gagggcataa tggacaatta aatctacatt 300
aattgaactc atttgggaag taaacaaaat ccatattctg gtgtaaatca aactatttga 360
tgcggattta ctaagatcct atgttaattt tagacatgac tggccaaagg tttcagttag 420
ttcatttgtc acggaaaggt gttttcataa gtccaaaact ctaccaactt ttttgcacgt 480
catagcatag atagatgttg tgagtcattg gatagatatt gtgagtcagc atggatttgt 540
gttgcctgga aatccaacta aatgacaagc aacaaaacct gaaatgggct ttaggagaga 600
tggtttatca atttacatgt tccatgcagg ctaccttcca ctactcgaca tggttagaag 660
ttttgagtgc cgcatatttg cggaagcaat ggcactactc gacatggtta gaagttttga 720
gtgccgcata tttgcggaag caatggctaa cagatacata ttctgccaaa ccccaagaag 780
gataatcact cctcttagat aaaaagaaca gaccaatgta caaacatcca cacttctgca 840
aacaatacac cagaactagg attaagccca ttacgtggct ttagcagacc gtccaaaaat 900
ctgttttgca agcaccaatt gctccttact tatccagctt cttttgtgtt ggcaaactgc 960
ccttttccaa ccgattttgt tcttctcacg ctttcttcat aggctaaact aacctcggcg 1020
tgcacacaac catgtcctga accttcacct cgtccctata aaagcccatc caaccttcac 1080
aatctcatca tcacccacaa caccgagcac cccaatctac agatcaattc actgacagtt 1140
caccgaggga tccatggcta agcggttagt cctctttgtg gcggtagtcg tcgccctcgt 1200
ggctctcacc gtcgctgaag gtgaggcctc tgagcaacta cagtgtgagc gcgagctcca 1260
ggagctccag gagcgcgagc tcaaggcatg ccagcaggtc atggaccagc agctccgaga 1320
cattagcccc gagtgccacc ccgtcgtcgt cagcccggtc gcgggacaat acgagcagca 1380
aatcgtggtg ccgcccaagg gcggatcttt ctaccccggc gagaccacgc caccgcagca 1440
actccaacaa cgtatatttt ggggaatacc tgcactacta aaaaggtatt acccaagtgt 1500
aacttgtccg cagcaggttt catactatcc aggccaagct tctccgcaac ggccaggaca 1560
aggtcagcag ccaggacaag ggcaacaatc aggacaagga caacaagggt actacccaac 1620
ttctccgcaa cagccaggac aatggcaaca accggaacaa gggcaaccag ggtactaccc 1680
aacttctccg cagcagccag gacaattgca acaaccagca caagggcagc aaccaggaca 1740
aggacaacaa ggtcggcagc caggacaagg gcaaccaggg tactacccaa cttcttcgca 1800
gctgcagcca ggacaattgc aacaaccagc acaagggcaa caagggcagc aaccaggaca 1860
agggcaacaa ggtcaacagc caggacaagg gcaacaacca ggacaaggac aacaaggtca 1920
acagccagga caagggcaac aaccaggaca agggcaacaa ggtcagcagc tcggacaagg 1980
acaacaaggg tactacccaa cttctctgca acagtcggga caagggcaac cagggtacta 2040
cccaacttct ctgcagcagc taggacaagg gcaatcaggg tactacccaa cttctccgca 2100
gcaaccagga caagggcagc agccaggaca attgcaacaa ccagcacaag ggcagcaacc 2160
agaacaaggg caacaaggtc agcagccagg acaagggcaa caaggccagc agccaggaca 2220
agggcagcaa ccgggacaag ggcaaccagg gtactaccca acttctccgc agcagtcagg 2280
acaagggcaa ccagggtact acccaacttc ttcgcagcag ccaacacaat cgcagcaacc 2340
aggacaaggg caacaaggtc agcaggtagg acaagggcaa caagctcagc agccaggaca 2400
agggcagcaa ccgggacaag ggcagccagg gtactaccca acttctccgc tgcagtcagg 2460
acaagggcaa ccagggtact acctaacttc tccgcagcag tcaggacaag ggcagcagcc 2520
aggacaattg caacaatcag cacaagggca aaaaggacag caaccaggac aaggtcaaca 2580
gccagggcaa gggcaacaag gtcagcagcc aggacaaggg caacaaggtc agcaaccggg 2640
gcaagggcag ccagggtact acccaacttc tccgcagcaa tcaggacaag ggcaacagcc 2700
aggacaatgg caacaaccag gacaagggca accaggatac tacccaactt ctccgttgca 2760
gccaggacaa gggcaaccag ggtacgaccc aacttctccg caacagccag gacaagggca 2820
gcaaccagga caattgcaac aaccagcaca agggcaacaa gggcagcaac tagcacaagg 2880
gcaacaaggg cagcaaccag cacaagtgca acaagggcag cagccagcac aagggcaaca 2940
aggtcagcag ctaggacaag ggcaacaagg tcagcagcca ggacaagggc agcaaccagc 3000
acaagggcaa caaggtcagc agccaggaca agggcaacaa ggtcagcagc caggacaagg 3060
gcagcaaccg ggacaagggc agccatggta ctacccaact tctccgcagg agtcaggaca 3120
agggcaacag ccaggacaat ggcaacaacc aggacaatgg caacaaccag gacaagggca 3180
accagggtac tacctaactt ctccgttgca gctaggacaa gggcaacaag ggtactaccc 3240
aacttctctg caacaaccag gacaagggca gcaaccagga caatggcaac aatcgggaca 3300
agggcaacat gggtactacc caacttctcc gcagctgtca ggacaagggc aacggccagg 3360
acaatggctg caaccaggac aagggcaaca agggtactac ccaacttctc cgcaacagtc 3420
aggacaaggg caacaactag gacaatggct gcaaccagga caagggcaac aagggtacta 3480
cccaacttct ctgcaacaga caggacaagg gcagcaatca ggacaagggc aacaaggcta 3540
ctacagctca taccatgtta gcgtggagca ccaggcggcc agcctaaagg tggcaaaggc 3600
gcagcagctc gcggcacagc tgccggcaat gtgccggctg gagggcggcg acgcattgtc 3660
ggccagccag tgatag 3676
<210>14
<211>20
<212>DNA
<213>Artificial Sequence
<400>14
tcggccagcc agtgataggg 20
<210>15
<211>20
<212>DNA
<213>Artificial Sequence
<400>15
tacgcaaacc gcctctcccc 20

Claims (10)

1.1Dx2m1 protein, which is obtained by mutating serine at position 118 of 1Dx2 protein amino acid sequence to cysteine;
the 1Dx2 protein is a protein shown in a) or b) as follows:
a) the amino acid sequence is a protein shown in a sequence 4;
b) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 4.
2. The protein of claim 1, wherein: the 1Dx2m1 protein is a protein shown in the following c) or d):
c) the amino acid sequence is a protein shown in a sequence 8;
d) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 8.
3. The biomaterial related to the 1Dx2m1 protein of claim 1 or 2, being any one of the following a1) to a 8):
A1) a nucleic acid molecule encoding the 1Dx2m1 protein of claim 1;
A2) an expression cassette comprising the nucleic acid molecule of a 1);
A3) a recombinant vector comprising the nucleic acid molecule of a 1);
A4) a recombinant vector comprising the expression cassette of a 2);
A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);
A6) a recombinant microorganism comprising the expression cassette of a 2);
A7) a recombinant microorganism comprising a3) said recombinant vector;
A8) a recombinant microorganism comprising the recombinant vector of a 4).
4. The related biological material according to claim 3, wherein: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):
1) the coding sequence is a cDNA molecule or a DNA molecule shown in a sequence 7;
2) a cDNA molecule or genomic DNA molecule having 95% or more identity to the nucleotide sequence defined in 1) and encoding the 1Dx2m1 protein of claim 1;
3) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and encodes the 1Dx2m1 protein of claim 1.
5. Use of the protein of claim 1 or 2 or the related biomaterial of claim 3 or 4) in any one of the following B1) -B10):
B1) regulating and controlling the processing quality of the wheat flour;
B2) regulating and controlling the baking quality of the wheat flour;
B3) regulating and controlling the gluten property and/or gluten strength and/or gluten quality of the wheat flour;
B4) regulating the dough rheological property and/or the extensibility and/or the stretch resistance and/or the kneading resistance of the wheat flour;
B5) regulating and controlling the SDS-sedimentation value of the wheat flour;
B6) regulating and controlling the glutenin swelling index of the wheat flour;
B7) regulating and controlling the water absorption and/or the dough forming time and/or the dough stabilizing time and/or the weakening degree in the flour quality instrument detection parameters of the wheat flour;
B8) regulating and controlling peak time and/or peak area and/or anti-fading value in the parameters of the kneading and mixing instrument of the wheat flour;
B9) cultivating transgenic wheat with improved flour processing quality and/or baking quality and/or gluten quality;
B10) and (5) wheat breeding.
6. A transgenic wheat having improved flour processing quality and/or baking quality and/or gluten quality, comprising the step of increasing the content and/or activity of the 1Dx2m1 protein of claim 1 in a recipient wheat to obtain a transgenic wheat; the transgenic wheat has a higher flour processing quality and/or baking quality and/or gluten quality than the recipient wheat.
7. The method of claim 6, wherein: the transgenic wheat has higher flour processing quality and/or baking quality and/or gluten quality than the acceptor wheat as shown in any one of C1-C9):
C1) the SDS-sedimentation value of the transgenic wheat flour is higher than that of receptor wheat;
C2) the swelling index of glutenin of the transgenic wheat flour is higher than that of receptor wheat;
C3) the water absorption rate in the detection parameters of the flour quality instrument of the transgenic wheat flour is higher than that of receptor wheat;
C4) the dough forming time in the flour quality instrument detection parameters of the transgenic wheat flour is longer than that of receptor wheat;
C5) the dough stability time in the flour quality instrument detection parameters of the transgenic wheat flour is longer than that of receptor wheat;
C6) the attenuation degree of the detection parameters of the flour quality instrument of the transgenic wheat flour is lower than that of receptor wheat;
C7) the peak time in the detection parameters of the kneading and mixing instrument of the transgenic wheat flour is higher than that of receptor wheat;
C8) the peak value area in the detection parameters of the kneading and mixing instrument of the transgenic wheat flour is higher than that of receptor wheat;
C9) the anti-fading value of the parameters detected by the kneading and mixing instrument of the transgenic wheat flour is lower than that of receptor wheat.
8. The method according to claim 6 or 7, characterized in that: the method for increasing the content and/or activity of the 1Dx2m1 protein in the receptor wheat is to overexpress the 1Dx2m1 protein in the receptor wheat.
9. The method of claim 8, wherein: the method for over-expressing the 1Dx2m1 protein of claim 1 in recipient wheat is to introduce the gene encoding the 1Dx2m1 protein of claim 1 into recipient wheat.
10. The method of claim 9, wherein: the nucleotide sequence of the coding gene of the 1Dx2m1 protein is a DNA molecule shown in a sequence 7.
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